The goal of this field is to attain a complete molecular and physiological understanding of intracellular oxidative stress. Workers seek to determine how reactive oxygen species are formed inside cells, which biomolecules they most rapidly damage, and how cells defend themselves against them. These problems may be most tractable in Escherichia coli. This model organism provides unique advantages for these studies, including the ability to generate hypersensitive mutants in the absence of oxygen. A mutant strain that cannot scavenge hydrogen peroxide has pushed work forward on several fronts. Because it releases endogenous H2O2 into the growth medium, one can quantify the rate at which H2O2 is generated inside aerobic cells. One can also easily impose low doses of H2O2 for an extended period of time, an approach that has revealed the cellular processes that are most sensitive to impairment by H2O2. Finally, by knocking out candidate genes, one can identify those that are critical in defending E. coli against micromolar H2O2 stress. In this application we propose to extend these studies by pursuing four aims: (1) To pinpoint the redox enzymes that most rapidly generate H2O2 inside E. coli. (2) To reveal the mechanism by which H2O2 and superoxide inactivate transketolase, which appears to be extremely vulnerable to inactivation. (3) To investigate how intracellular manganese protects E. coli against H2O2 stress. (4) To identify mechanisms that protect iron-sulfur enzymes from oxidants. Most aspects of the biochemistry of oxidative stress are conserved among all organisms. Most defensive strategies are widely distributed, too. Therefore, this investigation should shed light upon the molecular bases of obligate anaerobiosis, the killing mechanisms of phagocytes, and the nature and severity of endogenous oxidative stress.